CX-4945, an Orally Bioavailable Selective Inhibitor of ... · CX-4945, an Orally Bioavailable...

Therapeutics, Targets, and Chemical Biology

CX-4945, an Orally Bioavailable Selective Inhibitor of ProteinKinase CK2, Inhibits Prosurvival and Angiogenic Signalingand Exhibits Antitumor Efficacy

Adam Siddiqui-Jain, Denis Drygin, Nicole Streiner, Peter Chua, Fabrice Pierre, Sean E. O’Brien, Josh Bliesath,Mayuko Omori, Nanni Huser, Caroline Ho, Chris Proffitt, Michael K. Schwaebe, David M. Ryckman,William G. Rice, and Kenna Anderes

AbstractMalignant transformation and maintenance of the malignant phenotype depends on oncogenic and non-

oncogenicproteins that are essential tomediate oncogene signaling and to support the alteredphysiologic demandsinduced by transformation. Protein kinase CK2 supports key prosurvival signaling pathways and represents aprototypical non-oncogene. In this study, we describe CX-4945, a potent and selective orally bioavailable smallmolecule inhibitor of CK2. The antiproliferative activity of CX-4945 against cancer cells correlated with expressionlevels of the CK2a catalytic subunit. Attenuation of PI3K/Akt signaling by CX-4945 was evidenced by depho-sphorylation of Akt on the CK2-specific S129 site and the canonical S473 and T308 regulatory sites. CX-4945 causedcell-cycle arrest and selectively induced apoptosis in cancer cells relative to normal cells. Inmodels of angiogenesis,CX-4945 inhibited human umbilical vein endothelial cell migration, tube formation, and blocked CK2-dependenthypoxia-induced factor 1 alpha (HIF-1a) transcription in cancer cells.Whenadministeredorally inmurinexenograftmodels, CX-4945waswell tolerated anddemonstrated robust antitumor activitywith concomitant reductionsof themechanism-based biomarker phospho-p21 (T145). The observed antiproliferative and anti-angiogenic responses toCX-4945 in tumor cells and endothelial cells collectively illustrate that this compound exerts its antitumor effectsthrough inhibition of CK2-dependent signaling inmultiple pathways. Finally, CX-4945 is the first orally bioavailablesmall molecule inhibitor of CK2 to advance into human clinical trials, thereby paving the way for an entirely newclass of targeted treatment for cancer. Cancer Res; 70(24); 10288–98. �2010 AACR.

Introduction

Cancer is a disease of cellular dysregulation arising from anaccumulation of genetic errors and overexpression of elementsrequired to maintain the transformed state (1, 2). Mutationsarising in receptor tyrosine kinases and serine/threoninekinases are frequently implicated in oncogenesis and haveconsequently been the focus of extensive efforts to designmolecularly targeted anticancer therapies (3). Other proteins,including kinases, that may not be directly transformativebut serve to sustain the cancer phenotype have been lessconsidered as anticancer targets until recently. A new modelof cancer is emerging, whereby the establishment and main-tenance of malignancy depends on the cooperation of deregu-lated oncogenes and an array of equally essential deregulated

non-oncogenes (1, 4). Althoughoncogenes are critical for directtransformation, non-oncogenes are required for maintainingthe transformed phenotype. The overexpression or deregula-tion of typically nonmutated non-oncogene products protectsthe cell from the increased burden of cellular stress that is aninvariable hallmark of transformation resulting from onco-genic activation (1, 2, 4). As this overexpression of otherwisenormal proteins is required to maintain oncogenic signaling,inhibition of non-oncogenic signaling represents an under-exploited therapeutic approach for targeting processes essen-tial for maintaining the cancer phenotype.

Among the proteins that sustain the transformed pheno-type, protein kinase CK2 has recently become recognized as aprototypical non-oncogene (5). Protein kinase CK2 is a tetra-meric enzyme composed of 2 catalytic (a and/or a0) subunitsand 2 regulatory (b) subunits. CK2 is a highly conserved,constitutively active serine/threonine protein kinase thatregulates multiple pathways including PI3K/Akt and WNTsignaling cascades, NF-kB transcription, and the DNA damageresponse (reviewed in refs. 6 and 7). CK2 can be described as apromoter of the hallmarks of cancer, further emphasizing theknown pleiotropy of this kinase. Although CK2 has long beenknown for its proproliferative and anti-apoptotic properties,an understanding of the extensive contribution of this proteinkinase to tumor maintenance is just beginning to emerge (5).

Authors' Affiliation: Cylene Pharmaceuticals, San Diego, California

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Adam Siddiqui-Jain, Cylene Pharmaceuticals,5820 Nancy Ridge Drive, Suite 200, San Diego, CA 92121. Phone: 858-875-5121; Fax: 858-875-5101. E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-10-1893

�2010 American Association for Cancer Research.

CancerResearch

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In keeping with its non-oncogene profile, mutations in CK2have not been reported. However, CK2 gene amplificationsand an associated reduction in patient survival have beendescribed in squamous cell lung cancer (8). Likewise, CK2activity and expression levels are elevated in many cancers ofdiverse genetic background including breast (9), lung (8),prostate (10), colorectal (11), renal (12), and leukemias (13),and the overexpression of CK2 in cancer tissues has frequentlybeen linked to disease progression and poor prognosis(reviewed in refs. 14 and 6). Collectively, these characteristicsidentify CK2 as a scientifically validated cancer target thatremains unexploited as a therapeutic target.Non-oncogenic proteins fall into 2 broad categories: intrin-

sic,which principally regulate prosurvival processes within thecancer cell, and extrinsic, which develop and support thetumor microenvironment (1). CK2 may be described as pro-moting both intrinsic and extrinsic non-oncogenic processesas a consequence of its impact onmultiple signaling pathways.CK2 does not function simply as a signal transduction kinasein the classical sense, mediating "longitudinal signaling" fromthe cell surface to the nucleus, but rather plays a "lateral role"in multiple prosurvival signaling cascades (5, 6). These includeintrinsic processes such as the regulation of cell-cycle proteins,PI3K/Akt signaling, and apoptosis pathways (reviewed in refs.6, 7, and 15). The PI3K/Akt pathway is the most commonlyaltered signaling pathway in human cancers (16) and thispathway is susceptible to activation by CK2 (17, 18). Further-more, CK2 has been characterized as a "master regulator" ofangiogenesis (19), an extrinsic non-oncogene–driven process.CK2 is elevated under hypoxic conditions, regulates hypoxia-inducible transcription factor 1 alpha (HIF-1a) activity (20)and mediates the aberrant response of epithelial cells togrowth factor signaling (21). Thus, CK2 drives both intrinsicand extrinsic processes to promote the cancer phenotype.Several research groups have reported that inhibition of

CK2, either by siRNA directed toward CK2a (22) or by usingchemical CK2 inhibitors such as 4,5,6,7-tetrabromobenzotria-zole (23) and 2-(dimethylamino)-4,5,6,7-tetrabromo-1H-ben-zimidazole (24), induced apoptosis and prevented theproliferation of cancer cells. Furthermore, antisense oligonu-cleotides directed toward CK2a have been shown to induceapoptosis and exhibit antitumor activity in xenograft models(22, 25). Given the generalized overexpression of CK2 incancers, the central role of CK2 in multiple pathways thatsupport the cancer phenotype and the evidence that CK2 is anessential protein for cancer cell survival, the development ofselective CK2 inhibitors has emerged as an attractive targetedapproach for the treatment of cancers (26).Herein we describe CX-4945, a potent, selective, and ATP-

competitive inhibitor of both isoforms of the CK2 catalyticsubunits CK2a and CK2a0 with IC50 values of 1 nmol/L. CX-4945 inhibited prosurvival and angiogenic signaling in cancercell lines and exhibited antitumor activity in murine xeno-grafts. Collectively, these studies indicate that the antitumormechanism of action is mediated through inhibition of CK2-dependent intrinsic and extrinsic processes required to main-tain the cancer phenotype. Finally, CX-4945 is the first orallybioavailable small molecule inhibitor of CK2 to advance into

human clinical trials, thereby paving the way for an entirelynew class of targeted treatment for cancer.

Materials and Methods

MaterialsCX-4945 5-(3-chlorophenylamino)benzo[c][2,6]naphthy-

ridine-8-carboxylic acid) was synthesized by Cylene Pharma-ceuticals as a golden colored solid material (>99% pure byhigh-performance liquid chromatography assay). A 5 mmol/Lstock solution in dimethyl sulfoxide was prepared and storedat �70�C. The drug was diluted directly into growth mediaimmediately prior to use.

Enzyme assaysThe 238 kinase selectivity panel was conducted using the

Kinase Profiler service offered by Millipore, which utilizes aradiometric filter-binding assay. The percent inhibition ofeach kinase was estimated using 0.5 mmol/L CX-4945 atATP concentrations equivalent to the Km value for ATP foreach respective human recombinant kinase. The determina-tion of IC50 values was done at ATP concentrations equivalentto the Km for ATP for each kinase using 9 concentrations ofCX-4945 over a range of 0.0001 to 1 mmol/L. The Ki value(inhibition constant) for CX-4945 against recombinant CK2was determined by graphing the IC50 values of CX-4945determined in the presence of various concentrations ofATP against the concentration of ATP. The Ki value is equiva-lent to the Y-intercept according to the Cheng–Prussoffequation (ref. 27; Ki ¼ IC50/(1þ[ATP]/Km), where Ki is theinhibition constant and Km is the Michaelis constant.

Cell cultureSUM 149PT and SUM 190PT inflammatory breast cancer

cells were purchased from Asterand. Human umbilical veinendothelial cells (HUVEC) were purchased from Lonza Inc.. Allother cell lines were purchased from American Tissue CultureCollection. Cell lines were cultured according to the suppliers’recommendations.

Akt pathway analysisBT-474 (breast) or BxPC-3 (pancreatic) cancer or HUVEC

cells were treated with a range of concentrations of CX-4945for the durations stated in the figure legends. Cells (1–3� 106)were washed twice with cold PBS and lysed in 100 mL of lysisbuffer [RIPA Buffer purchased from Sigma; 1 mmol/L dithio-thrietol, 1 mmol/L phenylmethylsulfonyl fluoride, 1� SerineProtease Inhibitor Cocktail Set I purchased from Calbiochem;1� Phosphatase Inhibitor Cocktail Set II and Microcystin LR(1 nmol/L) purchased from Sigma]. Primary antibodies (PTEN,Akt-S129, p21-T145, and p21) were purchased from Abcam,and PTEN-S370, PTEN-S380, Akt, Akt-S473, and Akt-T308 werepurchased from Cell Signaling.

Alamar Blue cell viability assayVarious cell lines were seeded at a density of 3,000 cells per

well 24 hours prior to treatment, in appropriate media, andthen treated with indicated concentrations of CX-4945. Sus-pensions cells were seeded and treated on the same day.

Antitumor Mechanisms of the Selective CK2 Inhibitor CX-4945

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Following 4 days of incubation, Alamar Blue (20 mL, 10% ofvolume per well) was added and the cells were furtherincubated at 37�C for 4–5 hours. Fluorescence with excitationwavelength at 530–560 nm and emission wavelength at590 nm was measured.

CK2 levels in breast cancer cellsBreast cancer cells were plated in 15-cm dish format over-

night. Cells were collected at 50% confluence by scraping intoice-cold PBS. The RNAwas isolated from cells using RNeasy kit(QIAGEN) and relative levels of CK2a, CK2a0, CK2b, andb-actin mRNA were measured using Applied Biosystems’proprietary primers-probe sets. Analyses were performed ona 7900HT Real-Time PCR System (Applied Biosystems). Pro-tein levels of CK2a, CK2a0, CK2b, and b-actin were measuredby Western hybridization. Primary antibodies (CK2a0 andCK2b) were purchased from Santa Cruz Biotechnology,CK2b from Millipore, and b-actin from Sigma.

Cell-cycle analysisUntreatedandCX-4945–treatedBT-474andBxPC-3cellswere

harvested by trypsinization, washedwith ice-cold PBS, and fixedwith ice-cold 70% ethanol for 4 hours. Cells were washed withPBS and RNA was digested for 1 hour at 37�C in staining buffer(0.25 mg/mL RNase A, 0.025 mg/mL propidium iodide, in PBS).Cells were analyzedwith a BDLSR II flow cytometer, histogramsweregeneratedwithBDFACSDivaSoftwarev4.1.2,andcell-cyclephases analyzed with ModFit LT 3.1 SP3.

Caspase 3/7 activationCaspase 3/7 enzyme activity was evaluated over a range of

CX-4945 concentrations in multiple cell lines with Caspase-Glo assay kit (Promega). The luminescence of each sample wasmeasured in a plate-reading luminometer (SpectraMax M5;Molecular Devices) with parameters of 1-minute lag time and0.5-second/well read time. Caspase activity was normalized tocell number as measured by CyQUANT (Invitrogen).

In vitro models of angiogenesisHUVEC proliferation, migration and tube-formation experi-

ments were performed by Southern Research Institute. Experi-mental details are provided in the supplementary materials.

Hypoxia studiesBT-474 and BxPC-3 cells were plated in 10-cm dish format

overnight. On the following day, the cells were treated withCX-4945 in degassed CO2-independent media (Invitrogen)for 48 hours under normoxic or hypoxic (94% N2, 1% O2, and5% CO2) conditions. At the end of treatment, cells werecollected by scraping into ice-cold PBS. The RNA was iso-lated from cells using RNeasy kit (QIAGEN) and relativelevels of aldolase, pVHL, and p53 mRNA were measuredusing Applied Biosystems’ proprietary primer-probe sets.Analyses were performed on a 7900HT Real-Time PCRSystem (Applied Biosystems).

Luciferase reporter assayHeLa cells were transfected with pHIF1-Luc vector

(Panomics) using Lipofectamine 2000 reagent (Invitrogen).

Twenty-four hours after the transfection, cells were treatedwith a range of concentrations of CX-4945 for 20 hours undernormoxic or hypoxic (94% N2, 1% O2, and 5% CO2) condi-tions. At the end of treatment, HIF-1a transcriptional activ-ity was measured by luciferase assay. The resulting data werenormalized for cell number determined with CyQUANT(Invitrogen).

In vivo studies in animalsFemale immunocompromised mice CrTac:Ncr-Foxn1nu (5–

7 weeks old) were obtained from Taconic Farms. Animals weremaintained under clean room conditions in sterile filter topcages. Animals received sterile rodent chow and water adlibitum. All procedures were conducted in accordance with theInstitute for Laboratory Animal Research Guide: The Care andUse of Laboratory Animals.

Efficacy studiesXenografts were initiated by subcutaneous injection of

BxPC-3 cells into the right hind flank region of each mouseor BT-474 cells were injected into the mammary fat pad ofmice implanted with estrogen pellets. When tumors reached adesignated volume of 150-200 mm3, animals were randomizedand divided into groups of 9 to 10mice per group. CX-4945 wasadministered by oral gavage twice daily at 25 or 75 mg/kg for31 and 35 consecutive days for the BT-474 and BxPC-3 models,respectively. Tumor volumes and body weights were mea-sured twice weekly. The length and width of the tumor weremeasured with calipers and the volume calculated using thefollowing formula: tumor volume ¼ (length � width2)/2.Percent tumor growth inhibition (TGI) values were calculatedon the final day of the study for CX-4945–treated compared tovehicle-treated mice and were calculated as 100� {1� [(Trea-tedFinal day � TreatedDay 1)/(ControlFinal day � ControlDay 1)]}.The significance of the differences between the treatedversus vehicle groups were determined using 1-way ANOVA(Graphpad Prism).

Pharmacodynamic studiesXenografts bearing BxPC-3 tumors (300–400 mm3) were

randomized and divided into 3 groups of 5 mice. CX-4945 wasadministered by oral gavage twice daily at 25 or 75 mg/kg for 3days. Four hours after the final dose of CX-4945, the animalswere euthanized, and tumors were resected and fixed in 10%buffered formalin for 24 hours before transferring to 70%ethanol. Immunohistochemistry (IHC) for phospho-p21(T145) was performed at Vel-lab Research. Stained tumorsections were visualized on a Zeiss Axiovert 200 fluorescencemicroscope (Zeiss) and the degree of staining was quantifiedusing Axiovision40AV.4.5.0.0 software.

Results

CX-4945 is a potent and selective inhibitor of CK2activity

Utilizing molecular modeling techniques and medicinalchemistry, CX-4945 was rationally designed to be a lowmolecular weight (MW 350), ATP-competitive inhibitor of

Siddiqui-Jain et al.

Cancer Res; 70(24) December 15, 2010 Cancer Research10290



CK2(Fig. 1A). A full description of its discovery and synthesiswill be provided elsewhere. CX-4945 proved to be a potent andATP-competitive (Fig. 1B) inhibitor of recombinant humanCK2 (CK2a IC50 ¼ 1 nmol/L and Ki ¼ 0.38 nmol/L � 0.02;CK2a0 IC50 ¼ 1 nmol/L) and was found to be selective forCK2 when evaluated in a biochemical kinase screen. Using asingle concentration of 500 nmol/L (500-fold greater thanthe IC50 of CK2), only 7 of the 238 kinases tested wereinhibited by more than 90% and those 7 were furthersubjected to IC50 determination (Column 1, Table 1). It iswell understood that enzymatic potency determined in cell-free systems does not always translate into biological activ-ity, therefore, CX-4945 (10 mmol/L) was evaluated in relevantcell-based functional assays for FLT3, PIM1, and CDK1, andwas found to be functionally inactive against these kinases(data not shown).

Antiproliferative activity and CK2 expressionWhen profiled against a panel of diverse cancer cell lines,

CX-4945 exhibited a broad spectrum of antiproliferative activ-ity. COMPARE analysis (http://dtp.nci.nih.gov/docs/compare/compare_methodology.html) of these data (Fig. 1C) indicatedthat the breast cancer cell lines displayed the widest range ofsensitivity to CX-4945.

In addition to assessment of the antiproliferative activity, apanel of 11 breast cancer cell lines was further characterizedto determine potential predictors of sensitivity to CX-4945. Forthis panel of cell lines, mRNA and protein expression levels ofthe CK2 subunits were determined (representative Westernblot shown in Fig. 2). An apparent correlation was foundbetween sensitivity to CX-4945 and CK2a mRNA, and proteinlevels (Table 2) but no correlations were observed betweensensitivity to CX-4945 and either mRNA or protein levels of the

Figure 1. A, chemical structure ofCX-4945. B, ATP concentration-dependent inhibition of CK2a byCX-4945. C, antiproliferativeactivity against a panel of cancercell lines. The baseline representsthe mean EC50 across all cell lines(5.5 mmol/L), negative logEC50

values represent cell lines ofgreater sensitivity than average,and positive logEC50 valuesrepresent cell lines that are lesssensitive than average.

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CK2a0 catalytic subunit or the regulatory CK2b subunit (datanot shown). Furthermore, the PI3K/Akt or PTEN mutationalstatus for the breast cancer panel did not reveal any clearcorrelations with antiproliferative activity (Table 2).

CX-4945 suppresses PI3K/Akt signaling in cancer cellsGiven the prominent role of CK2 as a regulator of the PI3K/

Akt signaling pathway by direct phosphorylation of PTEN (28)and Akt itself (17), combined with the fact that CX-4945 was

shown to be inactive against key kinases within this pathway(Column 2, Table 1), we examined the effects of CX-4945 onCK2-regulated PI3K/Akt activity. For this analysis we chose 2models on the basis of their PI3K status: (1) BT-474 breastcancer cells that bear an activating PIK3CA mutation and (2)BxPC-3 pancreatic cells in which overexpression of the epi-dermal growth factor receptor activates wild-type PI3K signal-ing by heterodimerization with HER3 (29). Treatment ofBT-474 cells and BxPC-3 cells with CX-4945 led to rapiddephosphorylation of the CK2 phosphorylation site, Akt(S129) (Fig. 3A), which was accompanied by significant reduc-tions in the phosphorylation levels of both canonical regulatorysites Akt (T308) and Akt (S473). No significant reduction inPTEN phosphorylation at the CK2 phosphorylation sites, S370/S380, were observed at this 4-hour time point (data not shown)suggesting that CX-4945 suppresses PI3K/Akt signaling bydirectly blocking the phosphorylation of Akt at Serine 129by CK2 rather than through activation of PTEN.

Cell-cycle arrest and caspase 3/7 activationCK2 has been widely characterized as an important

regulator of the cell cycle and has been found to regu-late cell-cycle transition through multiple phases in a

Table 1. IC50 determinations for CX-4945 against selected kinases from the 238-kinase selectivity panel

Kinase IC50 (nmol/L) Kinase IC50 (nmol/L)

CK2a 1 PI3Kb >500CK2a0 1 PI3Kd >500DAPK3 17 PI3Kg >500FLT3 35 PDK1 >500TBK1 35 AKT1 >500CLK3 41 AKT2 >500HIPK3 45 AKT3 >500PIM1 46 mTOR >500CDK1/cyclin B 56 p70S6K >500

'

Figure 2. Protein levels of CK2 subunits in a panel of breast cancer celllines.

Table 2. Antiproliferative activity of CX2-4945, CK2a mRNA, protein levels, and mutational status of thePI3K pathway in a panel of breast cancer cell lines

Cell Line CX-4945 EC50 (mmol/L) CK2a mRNAa CK2a proteina PI3K pathway mutation

SKBr3 1.71 2.96 1.87 WTMDA-MB-453 2.36 2.57 2.42 PIK3CABT-474 2.64 2.45 1.33 PIK3CAZR-75-1 4.99 1.85 2.00 PTENMDA-MB-231 6.42 1.46 1.49 WTMDA-MB-468 8.30 1.04 1.83 PTENT47D 8.39 1.66 1.72 PIK3CAMCF 7 8.88 1.39 1.25 PIK3CAHs578T 13.12 1.00 1.00 WTMDA-MB-361 15.32 1.33 1.32 PIK3CAUACC-812 20.01 1.28 1.44 WT

amRNA and protein levels normalized to levels found in Hs578T cells.





cell-type–dependent manner (30–35). Activated Akt has beenreported to phosphorylate and thereby decrease the proteo-somal stability of the p21 and p27 cell-cycle inhibitor proteinsallowing cell-cycle progression (36, 37). Treatment of cells withCX-4945 resulted in reduced phosphorylation of p21 (T145)and increased levels of total p21 and p27. Interestingly, treat-ment of BT-474 cells with CX-4945 induced a G2/M cell-cyclearrest, whereas treatment of BxPC-3 cells caused arrest tooccur in G1, indicating that cell-cycle regulation by CK2 is cell-type dependent (Fig. 3B). Because both CK2 and its substratessuch as Akt, may protect cells from apoptosis by phosphor-ylating a wide range of proteins involved in the apoptoticresponse (35, 38–40), we examined levels of caspase 3/7

activity in a set of transformed and normal cell lines inresponse to CX-4945 treatment. All cancer cell linesresponded to the 24-hour treatment with CX-4945 by induc-tion of caspase 3/7 activity in a concentration-dependentmanner, whereas all normal cell lines failed to show adetectable change in caspase 3/7 activity at concentrationsas high as 100 mmol/L (Fig. 3C).

AngiogenesisNormoxia. CK2 has been described as a master regu-

lator of angiogenesis due to its role in multiple mole-cular events that contribute to angiogenesis (19). Thisincludes regulation of the PI3K/Akt pathway, which has a

Figure 3. A, effects of 4 hourexposure to CX-4945 on Akt andp21 phosphorylation in BT-474and BxPC-3 cells. B, cell-cyclearrest induced by CX-4945in BT-474 and BxPC-3 cells(24 hours). C, caspase3/7 activation in cancer cell linesand normal cells treated withCX-4945 (24 hours).

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well-characterized role in mediating the response of HUVECto growth factor signaling (41). Treatment of HUVEC withCX-4945 led to the rapid dephosphorylation of the CK2phosphorylation site Akt (S129) in addition to both cano-nical regulatory sites, Akt (T308) and Akt (S473) and thedownstream Akt effector, p21 (T145) (Fig. 4A). We alsoexamined PTEN phosphorylation in response to CX-4945treatment. We observed a reduction of phosphorylation atthe CK2-phosphorylation sites on PTEN (S370/S380) and aconcomitant reduction in total PTEN levels, but only after 24hour exposure to CX-4945. Next we measured the effects ofCX-4945 on the ability of HUVEC to proliferate, migrate, andform tube-like structures as a result of serum stimulation invitro. CX-4945 inhibited HUVEC proliferation (IC50 ¼ 5.5mmol/L at 72 hour; data not shown), migration (IC50 ¼ 2mmol/L at 24 hour), and tube formation (IC50 ¼ 4.0 mmol/Lat 18 hour; Fig. 4B).

Hypoxia. Hypoxia, a condition often found in solidtumors, is known to increase CK2 activity leading to theupregulation of histone deacetylases (HDAC1 and HDAC2)that inhibit the expression of tumor-suppressor genes pVHLand p53, both of which control the activity of HIF-1a, a majorregulator of proangiogenic signaling (20, 42, 43). Therefore, weinvestigated the effects of CK2 inhibition on HIF-1a–depen-dent transcription under hypoxic conditions. Using qRT-PCR(quantitive real-time PCR), we demonstrated that exposure ofBT-474 and BxPC-3 cells to hypoxia for 48 hours resulted in theactivation of HIF-1a transcriptional activity, as indicated by a2- to 3-fold increase in levels of aldolase mRNA, a known targetof HIF-1a transcription. Moreover, hypoxia reduced the levelsof pVHL (2- to 3-fold) and p53 (2-fold). Treatment of BT-474and BxPC-3 cells with CX-4945 under hypoxic conditionsprevented downregulation of p53 and pVHL and reducedactivation of HIF-1a transcription as measured indirectlyby a reduction of aldolase expression (Fig. 4C). These datawere corroborated by the results obtained with a pHIF-1a/Lucreporter system in which CX-4945 inhibited hypoxia-activatedHIF-1a–driven expression of luciferase in a concentration-dependent manner (Fig. 4D).

Antitumor efficacy of CX-4945Antitumor efficacy studies were conducted in BT-474 and

BxPC-3 xenograft models in which CX-4945 showed dose-dependent antitumor activity in both models when adminis-tered orally twice a day (Fig. 5A and B). In the BT-474 model,CX-4945 (25 or 75 mg/kg) exhibited 88% and 97% TGI,respectively, with 2 of 9 animals in each group showing morethan 50% reduction in tumor size compared with the initialtumor volume. In the BxPC-3 model, CX-4945 (75 mg/kg)showed 93% TGI with 3 animals having no evidence of tumorremaining at the end of the treatment period. Extensivepreclinical and clinical pharmacokinetic evaluation of CX-4945 has been performed and will be described in a separatepublication. In brief, the peak plasma concentration (Cmax) ofCX-4945 was 15 mmol/L occurring at 15 minute postdose,which rapidly decreased to submicromolar levels (Clearance,CLs ¼ 2.7 L/kg/h), suggesting that efficacy was driven by Cmax

rather than sustained exposure above a threshold. CX-4945

was well tolerated at all doses tested as indicated by minimalbody weight loss and no overt toxicity.

Pharmacodynamic studiesCX-4945 was evaluated for its effect on p21 (T145) in BxPC-3

xenograft tissue using immunohistochemical methods. Thedata shown represent scoring derived from 4 representative,random images taken from each of 3 tumors per group(Fig. 5C). When compared to the vehicle group, the 25 and75 mg/kg groups showed dose-dependent reductions in thephosphorylation of p21 (T145) consistent with the antitumoreffects observed with these dose levels (representative imagesshown in Fig. 5C).

Discussion

In the reported studies we describe a novel, orally bioavail-able inhibitor of protein kinase CK2 and the consequences ofinhibiting this enzyme in a variety of biological assays. Wehave shown that CX-4945 modulated PI3K/Akt signaling, cell-cycle progression, regulatory CDK proteins p21 and p27, andultimately apoptosis in cancer cells. Our findings are consis-tent with the results obtained with the relatively nonselectivenatural product CK2 inhibitors apigenin and emodin, whichhave previously been reported to increase levels of p21 andp27, induce cell-cycle arrest, and trigger apoptosis in cancercells (44, 45). CK2 has been shown to regulate PI3K/Aktsignaling at multiple junctions along the signaling cascadeincluding the Akt protein (reviewed in ref. 6). The phosphor-ylation of Akt by CK2 (at residue S129) has been reported tohyperactivate Akt beyond the activity level achieved by thephosphorylations of the canonical regulatory sites, T308 andS473 (17, 46). We have shown that CX-4945 treatment of theBT-474 and BxPC-3 cancer cell lines resulted in significantreductions in the phosphorylation of Akt (S129) with corre-sponding decreases in the phosphorylation of the canonicalregulatory sites Akt (T308) and Akt (S473). Although the extentof modulation of Akt (T308) and Akt (S473) compared withAkt (S129) dephosphorylation varied between cell lines, theseresults highlight the specificity of Akt (S129) phosphorylationby CK2 and suggest that cell-type–specific factors influencethe degree to which Akt (S129) and hence CK2 regulates Aktsignaling. Moreover, modulation of Akt (S129) may serve as avaluable mechanism of action biomarker to determine theextent of pharmacodynamic modulation of CK2. Although theantibody reagents and methods to detect Akt (T308) and Akt(S473) are available and used in the clinical setting, validationof Akt (S129) antibodies, IHC and other clinically relevantdetection methods are under development.

CK2 has been described as a master regulator of angiogen-esis (19). Indeed, chemical inhibitors of CK2 have previouslybeen shown to inhibit proangiogenic activity in HUVEC andblock retinal neovascularization in mice (19, 21, 47). PI3K/Aktsignaling is known to regulate the response of endothelial cellsto growth factor signaling including proliferation, migration,and tube formation (41), and thus represents a molecularmechanism through which CK2 may drive angiogenic signal-ing. CX-4945 inhibited HUVEC proliferation, migration, and





Figure 4. A, effects of CX-4945 onproangiogenic signaling undernormoxia and hypoxia. Westernblots show the effects of CX-4945on Akt phosphorylationin HUVEC. B, micrographs showthe effects of CX-4945 on the tubeformation and migration in HUVEC(24 hours). C, effects of CX-4945on aldolase, pVHL, and p53expression in BT-474 and BxPC-3cells under hypoxic versusnormoxic conditions (48 hours).D, effects of CX-4945 onHIF-1a–driven luciferaseexpression in HeLa cells underhypoxic versus normoxicconditions (48 hours).

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tube formation. The treatment of HUVEC with CX-4945 led torapid dephosphorylation of Akt (S129) and also Akt (T308) andAkt (S473). Dephosphorylation of the CK2 phosphorylationsites on PTEN (S370/S380) was observed only after 24 hourexposure to CX-4945 with concomitant reductions in totalPTEN levels. Neither of the CK2 phosphorylation sites onPTEN or the levels of total protein were significantly affectedafter 4-hour treatment, yet dephosphorylation of Akt wasreadily observed, suggesting that the effects of CX-4945 on

Akt activity are mechanistically and temporally independentof CK2-regulated PTEN activity.

Another mechanism by which CK2 may contribute toangiogenesis is via regulation of HIF-1a. Hypoxia, a conditionthat is often present in solid tumors, stimulates HIF-1a andincreases the activity of CK2, therebymediating proangiogenicresponses required for adaptation to hypoxia (20, 42, 43).Elevated CK2 activity results in the upregulation of HDAC1/HDAC2 histone deacetylases that inhibit the expression of

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(Breast)

Figure 5. A and B, antitumorefficacy of CX-4945 in BT-474(breast) and BxPC-3 (pancreatic)xenograft models, respectively.Vehicle (sodium phosphate buffer)or CX-4945 was administered byoral gavage twice a day (bid).Tumor volumes are expressed asmean � SEM. *, P � 0.001,significant difference from thevehicle groups as determinedusing 1-way ANOVA.C, quantitation of IHC stainingfor P-p21 (T145) in BxPC-3xenografts with representativeimages.





tumor-suppressor genes pVHL and p53, both of whichdownregulate the activity of HIF-1a (20, 42, 43). CX-4945inhibited hypoxia-driven HIF-1a activity in cancer cells andprevented downregulation of pVHL and p53, potentiallyindicating that CX-4945 inhibits CK2 regulation of histonedeacetylases. Collectively these data demonstrate thatCX-4945 inhibits angiogenic signaling in HUVEC and inhibitsHIF-1a transcription under hypoxia, suggesting that CK2inhibition effectively disrupts important adaptive processesrequired to promote the tumor microenvironment.The hypothesis that tumors rely on CK2 for survival was

tested in murine xenografts. BT-474 breast cancer and BxPC-3 pancreatic xenografts were selected for antitumor efficacystudies. CX-4945 exhibited robust antitumor activity includ-ing partial and complete regressions. In vivo administrationof CX-4945 resulted in decreased phosphorylation of the Aktsubstrate p21 (T145) in tumor xenograft tissue demonstrat-ing a clear pharmacodynamic effect in mice. CX-4945 waswell tolerated as indicated by minimal body weight loss andno evidence of overt toxicity.The network of proproliferative signaling pathways sup-

ported by CK2 can be best described within the context of"intrinsic" and "extrinsic" non-oncogene–driven processes.Herein, we have characterized CX-4945 as an inhibitor ofboth "intrinsic" and "extrinsic" CK2-driven mechanisms anddemonstrated that CX-4945 is efficacious and well tolerated inxenograft models. Furthermore, we have shown that sensitiv-

ity to CX-4945 correlates with the expression levels of CK2a ina panel of breast cancer cell lines suggesting a potentialstrategy for patient selection. We have also demonstratedthat phospho-p21 (T145), serves as an effective mechanism ofaction biomarker for CK2 inhibition in vitro and in vivo, andmay have immediate clinical relevance because there is anestablished relationship between Akt activation, phospho-p21(T145) status, and clinical prognosis in breast cancers (48, 49).The pleiotropy and widespread distribution of CK2 in normaltissues have raised concerns that inhibition of CK2 might giverise to unacceptable toxicity. In practice, however, we haveshown that the consequences of CK2 inhibition are contextdependent and cancer cells are more vulnerable to CK2inhibition than normal cells. CX-4945 represents the firstorally bioavailable small molecule inhibitor of CK2 withacceptable pharmaceutical properties to warrant advance-ment into human clinical trials, thereby opening the doorto an entirely new class of therapeutics for cancer patients.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 05/27/2010; revised 08/09/2010; accepted 08/29/2010; publishedOnline 12/15/2010.

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2010;70:10288-10298. Cancer Res Adam Siddiqui-Jain, Denis Drygin, Nicole Streiner, et al. and Exhibits Antitumor EfficacyKinase CK2, Inhibits Prosurvival and Angiogenic Signaling CX-4945, an Orally Bioavailable Selective Inhibitor of Protein

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